水稻叶绿体基因文库的构建和精细限制图谱的制作

Construction of Cosmid Library and Detailed Physical Map of Rice Chloroplast

水稻幼叶在加有高浓度抗坏血酸的缓冲液中匀浆,以获得完整的叶绿体,从中分离到ctDNA得率高达100μg/100g叶,纯度足以用于限制性核酸内切酶分析。ctDNA经Mbo I部分酶解得到的片段克隆到载体pcos 2 EMBL的Bam HI位点,重组DNA经体外包装后感染宿主菌,筛选表型Tc^5Km^R的重组子,通过计数克隆有效率达5×10~4重组菌落/1微克插入DNA。用λ-末端酶对重组环状双链DNA在cos位点切成线性分子,产生两个(ON-L及ON-R)可供标记和杂交的末端,线性Cosmid DNA经限制酶部分消化,凝胶电泳分离,干燥凝胶放射自显影,得到了6种限制性核酸内切酶的限制图谱。水稻ctDNA全长为129.5kb,在ctDNA上Pvu Ⅱ、Sal Ⅰ、Pst Ⅰ、Hind Ⅲ、Eco RI及Bam HI的切点分别为11、12、17、37、67和44个,1R A和B为21.7kb,LSC为73.7kb,SSC为12.4kb。

The intact rice chloroplast was isolated by homogenizing rice leaves in a buffer containing asorbic acid of high concentration and centrifuging. The obtained rice chloroplast DNA (ctDNA) is in high yield (100 f33g/100g leaves) and pure enough for restriction endo-nuclease analysis.The ctDNA fragments generated by partial digestion with suitable restriction endonucle-ases were inserted into the vector pcos2 EMBL, and the recombinant DNAs were packaged in vitro and transfected the host bacteria cells. The tetracycline-sensitive and kanamycine-resis-tant recombinants were screened and the cloning efficiency approached over 10 000f33g inserted DNA. The recombinant DNAs were linearizied, digested by lambda-terminase at cos site, partially digested by restriction endonucleases and hybridized with [γ-s2P] ATP-labeled o'igo-nucleotide lambda mapping probes. Then the recombinant DNAs were seperated by electropho-resis and a detailed restriction endonuclease map has been constructed from the autoradiog-rams. The rice ctDNA has a length of 129.5kb and 11, 12, 17, 37, 67 and 44 recognition sites of Pvu Ⅱ, Sal Ⅰ, Pst Ⅰ, Hind Ⅲ, EcoR. Ⅰ and BamH Ⅰ, respectively. The inserted repeat(IR) sequence has a length of 21.7 kb, the large single copy(LSC) is 73.7 kb and small single copy (SSC), 12.4kb.